Project Summary The ability of cells to respond to their microenvironment is mediated by multiprotein signaling complexes. A particularly important type of multiprotein signaling complex involves cell surface receptors that transmit signals upon binding protein ligands on other cells. A premier example of such a receptor is the T cell receptor (TCR)? CD3 complex, a cardinal receptor of the immune system that is essential for protective responses to microbes and cancers. The TCR?CD3 complex is composed of a diverse TCR?? heterodimer noncovalently associated with the invariant CD3 dimers CD3??, CD3??, and CD3??. The TCR mediates peptide?MHC (pMHC) recognition, while CD3 transduces activation signals to the T cell. However, the mechanism whereby TCR engagement by pMHC initiates signaling remains a mystery. Our goal is to understand the early events of TCR signaling by obtaining information on: 1) the architecture of the TCR?CD3 complex; and 2) possible allosteric changes in TCR dynamics upon binding pMHC that are relayed to CD3. These studies will be undertaken through a highly integrated project that combines multiple approaches, technologies, and investigators. We will study both an MHC class I-restricted TCR (A6) and an MHC class II-restricted TCR (MS2-3C8). A6 recognizes a viral peptide from HTLV-1 bound to HLA-A2; MS2-3C8 recognizes a self-peptide from myelin basic protein bound to HLA-DR4. Our Aims are: 1. Epitope mapping and NMR-based docking of the wild-type TCR? CD3 complex. Although the extracellular regions of CD3 interact with those of TCR, crystallization of TCR? CD3 complexes has been thwarted by the low affinity of TCR?CD3 interactions. We will employ NMR chemical shift perturbation and PRE to determine binding epitopes between CD3 and TCR. These data will be used to determine a structure for the wild-type TCR?CD3 complex. 2. Structural analysis of affinity-matured TCR? CD3 complexes by X-ray crystallography. We will attempt to overcome the weak association between TCR and CD3 ectodomains by in vitro directed evolution using yeast display to stabilize TCR?CD3 complexes for crystallization. Information on binding epitopes from NMR will be used to design TCR mutant libraries by focusing mutagenesis on regions that contact CD3. 3. Biological validation of the TCR?CD3 structure. We will validate TCR?CD3 interfaces identified by NMR or crystallography by evaluating the effects of structure- guided mutations in TCR?CD3 interfaces on complex assembly, signaling, and T cell development using cellular assays and retrogenic mice expressing mutated TCR and CD3 components. 4. Dynamics analysis of free and pMHC-bound states of TCR?? ectodomains. We will address the allosteric change hypothesis by carrying out solution NMR analysis of TCR ectodomains in free form and bound to pMHC, for both TCRs A6 and MS2-3C8. We will address the role of the lipid bilayer on TCR dynamics through MD simulations. Our combined NMR and MD results will generate a biophysical model of signaling through the TCR?CD3 complex whose basic features should apply to signaling through other multiprotein complexes and receptors.